Project Summary/Abstract Background: Deregulation of sphingolipid metabolism alters pancreatic cancer progression and has thus received appreciable attention as a potential target for development of novel therapeutics. Intracellular ceramide levels are imperative for regulation of cellular senescence, apoptosis, and cell cycle arrest in response to stress stimuli such as chemotherapeutics. Preliminary data from our lab indicate a prominent role for a gene that regulates ceramide synthesis and exosome biogenesis, in the maintenance of pancreatic tumor progression. Orthotopic implantation of murine pancreatic ductal adenocarcinoma (PDA) cell lines stably expressing shRNA targeting this gene into immunodeficient mice significantly reduced pancreatic tumor burden when compared to controls. In addition, our preliminary data also indicate that this gene regulates exosome secretion from pancreatic cancer cell lines. Based on our exciting preliminary data, I have designed a study to decipher the molecular mechanisms by which this gene modulates pancreatic tumorigenesis. Objective/Hypothesis: Given this gene?s complex function in regulation of both cancer cell exosome secretion and intracellular ceramide levels, I propose the following central hypothesis: The function of this gene in pancreatic cancer is multiplex, potentially playing distinct roles during initiation and progression. The overall objective of this study is to determine the mechanisms by which this gene controls pancreatic carcinogenesis. Specific Aims: The specific aims of this study are: Aim 1) Evaluate the function of this gene in normal murine pancreas and pancreatic neoplasia formation and progression Aim 2) Determine the mechanism(s) by which this gene regulates pancreatic tumor growth Aim 2A) Assess the ability of this gene to alter cellular activities downstream of ceramide signaling Aim 2B) Evaluate the ability of exosomes secreted via this gene?s pathway to modulate pancreatic tumor expansion. Study Design: The approach proposed herein rigorously determines the function of this gene during PDA formation and progression using loss of function studies in established mouse and cell culture models of PDA. I will ablate our gene of interest in a widely-used mouse model of pancreatic cancer to determine the ability of this gene to regulate pancreatic cancer formation and progression. In addition, I propose mechanistic assays using murine pancreatic cancer (MPDA) cell lines generated by our group which stably express constructs allowing knockdown and overexpression studies that will illuminate the effect of exosome secretion and regulation of intracellular ceramide levels on pancreatic carcinogenesis mediated by this gene. Impact: Pancreatic cancer is projected to become the 2nd leading cause of cancer- related death in the US by 2020. Given this notable projected increase in incidence, pancreatic cancer remains a major problem for overall human health. Consistent with the overall mission of the NIH National Cancer Institute, the proposed studies may identify new therapeutic targets critical for development of new strategies to prevent, treat, and cure patients.